Oncolytic virotherapy is a promising approach because the efficient transduction and cancer cell-specific viral replication can boost therapeutic efficacy. However, the large size of oncolytic viruses hinders their transvascular and interstitial movement, thus significantly limiting their delivery to cancer cells and anti- tumor efficacy. Our findings from the current funding period showed that cancer cell apoptosis produced void spaces and channel-like structures, which enhanced the initial delivery, viral spread and therapeutic efficacy of oncolytic herpes-simplex-virus (HSV) after intratumor injection. But in order to improve the systemic efficacy of oncolytic virotherapy targeted both at primary and metastatic tumors, it is essential to identify agents (e.g., cytotoxic agents) or strategies that will improve the vascular extravasation and intratumoral distribution of viral particles injected intravenously. In the proposed studies we will test the hypothesis that targeting tumor blood vessels will improve the delivery of viral vectors and the anti-tumor efficacy of oncolytic HSV. The tumor vasculature will be targeted with endothelial cell targeting agents (anti-integrin 14 or 15 blocking antibodies) or cytotoxics (cyclophosphamide, docetaxel) that target both endothelial and cancer cells. The agents selected are known to induce endothelial cell apoptosis and remodel the tumor vasculature. We will use multiphoton laser scanning microscopy to measure the vascular pore cutoff size of large particles, vascular remodeling (e.g. vessel density, diameter, perfusion), and virus extravasation and spread in human mammary carcinoma cells implanted in mammary fat pad windows in immunodeficient mice. Because of the short-half life of virus in blood (HSV ~ 3 hours), we will determine for each agent the peak-time of large particle extravasation. Based on the peak-time of particle extravasation, oncolytic HSV will be injected systemically and the fraction of vessels associated with infected cells will be determined. The correlation between endothelial apoptosis and particle extravasation will be assessed in tumor sections. We will test if the administration of endothelial targeting agents or cytotoxics before the intravenous injection of oncolytic virus will produce a longer tumor growth delay in mammary fat pad tumors and reduce spontaneous metastasis formation in the lung. We will also determine if the repeated administration (cycling) of cytotoxics or endothelial targeting agents with oncolytic HSV will improve the intratumoral distribution of large particles and the spread and therapeutic efficacy of oncolytic HSV. We expect that the results of these translational studies will identify single agents and / or approaches that will enhance the vascular extravasation and intratumoral spread of virus, and the efficacy of oncolytic virotherapy targeted at tumors.